We have recently discovered novel intron-derived long noncoding RNAs (lncRNAs) that are processed on both ends by the snoRNA machinery (we named these sno-lncRNAs). Most snoRNAs are processed from introns. After debranching, exonucleases degrade the introns but not the snoRNAs because these associate with specific factors that prevent degradation and lead to snoRNP assembly. Sno-lncRNAs are processed from introns that contain two snoRNAs. During exonucleolytic trimming, the sequences between the snoRNAs are not degraded, leading to the accumulation of lncRNAs lacking 5'cap structures or 3'poly(A) tails. Some of these RNAs can accumulate to very high levels in cells but were missed before because they are long, they derive from introns, and they lack poly(A) tails. These constitute an entirely new class of nuclear lncRNAs and this proposal is to study them in greater detail. Results so far suggest that whenever introns contain not one but two snoRNA genes, sno-lncRNAs can be produced. Such RNAs can be found in every cell examined so far, and appear to be widely expressed in tissues. Further, the genomic region encoding some of the most abundant of these RNAs (15q11-q13) is specifically deleted in an important human genetic disease, Prader-Willi Syndrome. We will characterize how sno-lncRNAs are made, what proteins associate with them and how many of them there are. In addition, we will ask what they do in cells and begin to address their possible role in Prader-Willi Syndrome. In the first aim, we will characterize the structure and expression of sno-lncRNAs. We will also examine their sub-nuclear localization, and efforts will be made to follow their synthesis and localization dynamically and throughout the cell cycle. Finally in this aim, we will carry out genomewide studies in several different cell types and using several different approaches in order to identify more sno-lncRNAs, some of which will be studied in greater detail.
The second aim i s to investigate the mechanism of action of sno-lncRNAs. Of particular interest is the connection of the chr15 sno-lncRNAs to Fox1 family splicing regulators. Compelling evidence suggests that these lncRNAs may bind Fox2 and perhaps Fox1 with high affinity. Thus, they may act as molecular "sponges" to titrate splicing factors since in preliminary experiments we have found that they alter splicing patterns. This angle will be examined in detail and could lead directly to new insights into Prader-Willi pathology. We will also ask whether some sno-lncRNAs can alter chromatin organization like a number of other lncRNAs do. Finally, since these new RNAs appear to be relatively stable and strictly retained in the nucleus, we will begin to use our knowledge of their expression to generate a new class of vectors for nuclear expression of a variety of sequences.

Public Health Relevance

We have discovered a new class of long nuclear retained noncoding RNAs called sno-lncRNAs. The RNAs are derived from introns and appear to be processed by machinery involved in the production of small nucleolar RNAs. Some of these new RNAs are associated with an important human disease, Prader-Willi Syndrome. This proposal is to learn more about how sno-lncRNAs are produced and how they act in cells.